Hollie I. Swanson

The AH-receptor: genetics, structure and function.

The AH-receptor is a ligand-activated transcription factor that regulates a number of biological responses to planar aromatic hydrocarbons. Interest in this receptor is related to its role in the toxic action of a variety of environmental chemicals, the simplicity and elegance of the murine genetics that led to its characterization and the distinctive mechanism by which this receptor activates gene expression. Recent cloning experiments have demonstrated that the AH-receptor is structurally related to the Per, ARNT and Sim proteins. Members of this newly described gene family are characterized by two N-terminal domains, the most characteristic of which is a motif referred to as a PAS domain. In the AH-receptor, this domain harbours sequences involved in the formation of a hydrophobic pocket that bind receptor agonists. Adjacent to the PAS domain in the AH-receptor, ARNT and Sim proteins is a basic/helix-loop-helix (bHLH) domain that appears to mediate heterodimerization and sequence specific DNA binding properties. The observation that the bHLH domain is present in the AH-receptor and the ARNT protein, a factor required for proper AH-receptor function, suggests that these proteins are heterodimeric partners that activate gene expression in a manner similar to Myc/Max and MyoD/E2A. The objectives of this review are to describe recent experimental results in this field and to use this information to develop a molecular model of AH-receptor mediated signal transduction.

DNA Binding Specificities and Pairing Rules of the Ah Receptor, ARNT, and SIM Proteins

The Ah receptor (AHR), the Ah receptor nuclear translocator protein (ARNT), and single-minded protein (SIM) are members of the basic helix-loop-helix-PAS (bHLH-PAS) family of regulatory proteins. In this study, we examine the DNA half-site recognition and pairing rules for these proteins using oligonucleotide selection-amplification and coprecipitation protocols. Oligonucleotide selection-amplification revealed that a variety of bHLH-PAS protein combinations could interact, with each generating a unique DNA binding specificity. To validate the selection-amplification protocol, we demonstrated the preference of the AHR•ARNT complex for the sequence commonly found in dioxin-responsive enhancers in vivo (TNGCGTG). We then demonstrated that the ARNT protein is capable of forming a homodimer with a binding preference for the palindromic E-box sequence, CACGTG. Further examination indicated that ARNT may have a relaxed partner specificity, since it was also capable of forming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG. Coprecipitation experiments using various PAS proteins and ARNT were consistent with the idea that the ARNT protein has a broad range of interactions among the bHLH-PAS proteins, while the other members appear more restricted in their interactions. Comparison of this in vitro data with sites known to be bound in vivo suggests that the high affinity half-site recognition sequences for the AHR, SIM, and ARNT are T(C/T)GC, GT(G/A)C (5′-half-sites), and GTG (3′-half-sites), respectively.

Polychlorinated Biphenyl-77 Induces Adipocyte Differentiation and Proinflammatory Adipokines and Promotes Obesity and Atherosclerosis

Background Obesity, an inflammatory condition linked to cardiovascular disease, is associated with expansion of adipose tissue. Highly prevalent coplanar polychlorinated biphenyls (PCBs) such as 3,3′,4,4′-tetrachlorobiphenyl (PCB-77) accumulate in adipose tissue because of their lipophilicity and increase with obesity. However, the effects of PCBs on adipocytes, obesity, and obesity-associated cardiovascular disease are unknown. Objectives In this study we examined in vitro and in vivo effects of PCB-77 on adipocyte differentiation, proinflammatory adipokines, adipocyte morphology, body weight, serum lipids, and atherosclerosis. Methods PCB-77 or 2,2′,4,4,5,5′-hexachlorobiphenyl (PCB-153) was incubated with 3T3-L1 adipocytes either during differentiation or in mature adipocytes. Concentration-dependent effects of PCB-77 were contrasted with those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For in vivo studies, we treated C57BL/6 wild-type (WT) or aryl hydrocarbon receptor (AhR)−/− mice with vehicle or PCB-77 (49 mg/kg, by intraperitoneal injection) and examined body weight gain. In separate studies, we injected ApoE−/− mice with vehicle or PCB-77 over a 6-week period and examined body weight, adipocyte size, serum lipids, and atherosclerosis. Results Low concentrations of PCB-77 or TCDD increased adipocyte differentiation, glycerol–3-phosphate dehydrogenase activity, and expression of peroxisome proliferator–activated receptor γ, whereas higher concentrations inhibited adipocyte differentiation. Effects of PCB-77 were abolished by the AhR antagonist α-naphthoflavone. PCB-77 promoted the expression and release of various proinflammatory cytokines from 3T3-L1 adipocytes. Administration of PCB-77 increased body weight gain in WT but not AhR−/− mice. ApoE−/− mice injected with PCB-77 exhibited greater body weight, adipocyte hypertrophy, serum dyslipidemia, and augmented atherosclerosis. Conclusions Our findings suggest that PCB-77 may contribute to the development of obesity and obesity-associated atherosclerosis.

DNA binding and protein interactions of the AHR/ARNT heterodimer that facilitate gene activation

Gene activation by the aryl hydrocarbon receptor (AHR) and its DNA binding partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) requires a number of sequential steps that occur following the binding of ligand and entry of the AHR into the nuclear compartment. This includes heterodimerization of the AHR and ARNT, formation of the appropriate amino acid/nucleotide contacts at the GCGTG recognition site and interactions between either the AHR or ARNT with proteins that facilitate changes in chromatin structure. The majority of these steps are likely modulated by changes in both phosphorylation and oxidation status of the AHR, ARNT and associated proteins. Studies of both the basic helix-loop-helix transcription factors and the nuclear hormone receptor family can provide significant insights into how this unique signaling pathway activates its target genes.

Coplanar polychlorinated biphenyls impair glucose homeostasis in lean C57BL/6 mice and mitigate beneficial effects of weight loss on glucose homeostasis in obese mice.

Background: Previous studies demonstrated that coplanar polychlorinated biphenyls (PCBs) promote proinflammatory gene expression in adipocytes. PCBs are highly lipophilic and accumulate in adipose tissue, a site of insulin resistance in persons with type 2 diabetes. Objectives: We investigated the in vitro and in vivo effects of coplanar PCBs on adipose expression of tumor necrosis factor α (TNF-α) and on glucose and insulin homeostasis in lean and obese mice. Methods: We quantified glucose and insulin tolerance, as well as TNF-α levels, in liver, muscle, and adipose tissue of male C57BL/6 mice administered vehicle, PCB-77, or PCB-126 and fed a low fat (LF) diet. Another group of mice administered vehicle or PCB-77 were fed a high fat (HF) diet for 12 weeks; the diet was then switched from HF to LF for 4 weeks to induce weight loss. We quantified glucose and insulin tolerance and adipose TNF-α expression in these mice. In addition, we used in vitro and in vivo studies to quantify aryl hydrocarbon receptor (AhR)-dependent effects of PCB-77 on parameters of glucose homeostasis. Results: Treatment with coplanar PCBs resulted in sustained impairment of glucose and insulin tolerance in mice fed the LF diet. In PCB-77–treated mice, TNF-α expression was increased in adipose tissue but not in liver or muscle. PCB-77 levels were strikingly higher in adipose tissue than in liver or serum. Antagonism of AhR abolished both in vitro and in vivo effects of PCB-77. In obese mice, PCB-77 had no effect on glucose homeostasis, but glucose homeostasis was impaired after weight loss. Conclusions: Coplanar PCBs impaired glucose homeostasis in lean mice and in obese mice following weight loss. Adipose-specific elevations in TNF-α expression by PCBs may contribute to impaired glucose homeostasis.

Mechanisms of anti-carcinogenesis by indole-3-carbinol. Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation.

The induction of oxidation and conjugation enzymes, the scavenging of carcinogen electrophiles, and the inhibition of aflatoxin B1 (AFB1) activation were examined as possible mechanisms of anti-carcinogenesis by indole-3-carbinol (I3C). Liver microsomal 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities were not induced significantly in rainbow trout fed diets containing 500-2000 ppm I3C for 8 days compared to trout fed the control diet. Furthermore, no detectable changes in the specific contents of cytochrome P-450 isozymes LM2 and LM4b, as measured by Western-blotting and immunoquantitation, were found in liver microsomes following dietary I3C administration. Dietary I3C had no significant effect on liver microsomal uridine diphosphate-glucuronyl-transferase activity, measured using the substrates 1-naphthol and testosterone, or on cytosolic glutathione S-transferase activity, measured using the substrate styrene oxide. The ability of I3C or its acid reaction products (RXM; generated by the reaction of I3C with HCl) to act as scavengers for the direct alkylating agent AFB1-8,9-Cl2 was examined. Addition of I3C or RXM to in vitro incubations did not inhibit the covalent binding of AFB1-8,9-Cl2 to calf thymus DNA. Kinetic analyses of microsome-mediated binding of AFB1 to DNA in vitro indicated that RXM inhibited the metabolic activation of AFB1. RXM increased the apparent Km for the AFB1-DNA binding reaction without changing the associated Vmax; the apparent Km values at 0, 3.5, 35, and 350 microM RXM were 35, 38, 66, and 86 microM for trout liver microsomes. RXM also inhibited the activation of AFB1 by rat liver microsomes, but I3C was not an effective inhibitor against AFB1-DNA binding mediated by either rat or trout liver microsomes. The results of the present study indicate that inhibition of microsome-activated AFB1 binding to DNA by I3C products may be of significant importance in I3C inhibition of hepatocarcinogenesis in trout and other species. The inhibition of carcinogen activation by I3C is contrasted with the mechanism of anti-carcinogenesis by beta-naphthoflavone, which involves induction of xenobiotic metabolizing enzymes.

Role of the xenobiotic receptor in inflammatory bowel disease

Background: Gene‐environment interplay modulates inflammatory bowel diseases (IBD). Dioxin‐like compounds can activate the aryl hydrocarbon receptor (AhR) and alter macrophage function as well as T‐cell polarization. We hypothesized that attenuation of the AhR signaling pathway will ameliorate colitis in a murine model of IBD. Methods: Dextran sulfate sodium (DSS) colitis was induced in C57BL/6 AhR null mice (AhR−/−), heterozygous mice (AhR−/+), and their wildtype (WT) littermates. Clinical and morphopathological parameters were used to compare the groups. Patients: AhR pathway activation was analyzed in biopsy specimens from 25 IBD patients and 15 healthy controls. Results: AhR−/− mice died before the end of the treatment. However, AhR−/+ mice exhibited decreased disease activity compared to WT mice. The AhR−/+ mice expressed less proinflammatory cytokines such as tumor necrosis factor alpha (TNF‐&agr;) (6.1‐ versus 15.7‐fold increase) and IL17 (23.7‐ versus 67.9‐fold increase) and increased antiinflammatory IL‐10 (2.3‐fold increase) compared with the AhR+/+ mice in the colon. Colonic macrophage infiltration was attenuated in the AhR−/+ group. AhR and its downstream targets were significantly upregulated in IBD patients versus control (CYP1A1 −19.9, and IL8‐ 10‐fold increase). Conclusions: Attenuation of the AhR receptor expression resulted in a protective effect during DSS‐induced colitis, while the absence of AhR exacerbated the disease. Abnormal AhR pathway activation in the intestinal mucosa of IBD patients may promote chronic inflammation. Modulation of AhR signaling pathway via the diet, cessation of smoking, or administration of AhR antagonists could be viable strategies for the treatment of IBD. (Inflamm Bowel Dis 2010;)

Mapping the Protein/DNA Contact Sites of the Ah Receptor and Ah Receptor Nuclear Translocator

The Ah receptor (AHR) and its DNA binding partner, the Ah receptor nuclear translocator (ARNT), are basic helix-loop-helix proteins distinguished by their PER, AHR, ARNT, and SIM (PAS) homology regions. To identify the amino acids of the AHRARNT heterodimer that contact the TNGCGTG recognition sequence, we have performed deletion mapping and amino acid substitutions within the N termini of both the AHR and ARNT. The ability of the variant AHR and ARNT proteins to bind DNA and activate gene transcription was determined by the gel shift analysis and transient transfection assays. We have found that the amino acids of ARNT that contact DNA are similar to those of other basic/helix-loop-helix proteins and include glutamic acid residue 83 and arginine residues 86 and 87. Although our initial experiments indicated that DNA binding of the AHR may involve two regions that are bordered by amino acids 9-17 and amino acids 34-42, further analysis demonstrated that only amino acids 34-39 are critical for the AHRTNGC interaction. These experiments indicate that while the structural features of the ARNTGTG complex may closely resemble that deduced for proteins such as Max, E47, and USF, the AHRTNGC complex may represent a unique DNA binding form of basic/helix-loop-helix proteins.

Activation of the aryl hydrocarbon receptor by TCDD inhibits senescence: a tumor promoting event?

Activation of the aryl hydrocarbon receptor (AHR) by the agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to promote tumor formation in both liver and skin. In the liver, but not the skin, the AHR-mediated events that contribute to TCDDs tumor promoting activities have been studied in some detail and are thought to involve perturbation of cell fate processes. However, studies performed using cultured cells have often resulted in apparent contradictory results indicating that the impact of TCDD on cell fate processes may be cell context dependent. We and others have shown that in primary cultured keratinocytes TCDD increases post-confluent proliferation and increases late differentiation. Further, our studies performed in these cells indicate that TCDD can also inhibit culture-induced senescence. While senescence, a permanent cell cycle arrest, is emerging as an important process regulated by oncogenes and considered to be of therapeutic importance, its role with respect to TCDD/AHR mediated tumor promotion has not been fully considered. The intent of this article is to focus primarily on senescence as a cell process relevant to skin tumorigenesis and explore the idea that the inhibition of senescence by TCDD could be an important mechanism by which it may exert its tumor promoting effects in the skin.

Lack of the Aryl Hydrocarbon Receptor Leads to Impaired Activation of AKT/Protein Kinase B and Enhanced Sensitivity to Apoptosis Induced via the Intrinsic Pathway

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that is best known for its role in mediating the toxicity of many environmental contaminants such as 2,3,7,8 tetrachlorodibenzo-p-dioxin. However, the endogenous role of AHR, especially with respect to the apoptotic process, is largely unknown and contradictory. In this report, we have used a mouse hepatoma cell line (Hepa1c1c7) and its AHR-deficient derivative (LA1) to examine the effect of differing AHR levels on apoptosis susceptibility, in particular, apoptosis regulated by the intrinsic pathway. Toward this end, the cells were subjected to UV irradiation, hydrogen peroxide, and serum starvation. Analyses of a number of different endpoints of apoptosis revealed that the LA1 cells were more sensitive to these stresses than the wild-type cells, indicating that the AHR plays a cytoprotective role in the face of stimuli that initiate the intrinsic apoptotic pathway. A direct role of the AHR in mediating this effect was confirmed using both pharmacological and molecular approaches. Further analyses imply that lack of the AHR leads to an impaired survival response mediated by phosphatidylinositol 3′-OH kinase-Akt/protein kinase B and, to a lesser degree, epidermal growth factor receptor activation. These findings indicate that exploring the use of the AHR antagonist as agents that enhance the proapoptotic actions of cancer therapies may be a valid approach.